Functional characterization of the novel centrosomal protein Nlp (ninein-like protein)

The centrosome is the major microtubule organizing centre (MTOC) in animal cells. Most microtubules (MTs) emanate from the centrosome, where gamma-tubulin ring complexes (gammaTuRCs) act as templates for MT nucleation. During interphase, the centrosome organizes a MT array that imparts shape and polarity to the cell and is essential for intracellular transport and positioning of organelles such as the Golgi apparatus. During mitosis, centrosomes ensure bipolarity and correct orientation of the spindle by forming the spindle poles. In order to switch from the interphasic to the mitotic state, the centrosome undergoes a structural reorganization, termed maturation, which is mainly characterized by an increase in MT nucleation activity. A full appreciation of how centrosomes contribute to cellular functions requires the isolation and characterization of unknown centrosome-associated molecules. Here we describe the identification and characterization of a novel centrosomal component, the human protein Nlp (ninein-like protein) related to the previously characterized MT-anchoring protein ninein. In the first part of the present thesis we describe the identification of Nlp as a novel centrosomal substrate of Polo-like kinase 1 (Plk1), an important regulator of mitosis whose activity is required for centrosome maturation. Nlp interacts with two distinct gammaTuRC components, gamma-tubulin and hGCP4, and stimulates MT nucleation. Plk1 phosphorylates Nlp and disrupts its centrosomal association. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites induces defects in mitotic spindle formation. We propose that Nlp acts as a gammaTuRC binding protein (GTBP), contributing to the MT nucleation activity of the centrosome during interphase. At the onset of mitosis, the displacement of Nlp from the centrosome triggered by Plk1 phosphorylation could represent an important step in the maturation process which allows the centrosome to switch from the interphasic to the mitotic state. Thus, we conclude that Nlp, as well as the related protein ninein, plays an important role in MT organization. However the function of these two proteins possibly diverged during evolution: whilst Nlp gained a more prominent role in MT nucleation, ninein became principally involved in MT anchoring. In the second part of this thesis we report the initial characterization of the molecular mechanisms underlying the ability of Nlp and ninein to induce the fragmentation of the Golgi apparatus when overexpressed in human cells. We show that the ability of these two centrosomal proteins to affect the organization of the Golgi clearly depends on their capacity to associate with the cytoplasmic dynein-dynactin complex, a molecular motor complex primarly involved in the maintainance of Golgi architecture. We propose that the excess of Nlp and ninein could induce the disruption of the Golgi apparatus by sequestering the dynein-dynactin complexes. Future investigations should be aimed at understanding whether the dissociation of the Golgi apparatus from the centrosome induced by the excess of Nlp and ninein could interfere with cell migration and cell polarization processes, which require a highly coordinated action of these two organelles. Cell migration and cell polarization represent critical events for immune responses as well as for embryonic development, invasive growth and metastasis. Thus, our findings raise the interesting possibility that an upregulation in the expression levels of structural centrosomal proteins could represent the molecular basis for developmental disorders and malfunctioning of the immune system and, on the other hand, modulate the acquisition of invasive properties by neoplastic cells.